(a) Technical Field
The present disclosure relates to a protein-active agent conjugate. The protein (e.g., an oligopeptide, a polypeptide, an antibody, or the like) has substrate specificity for a desired target, and the active agent (e.g., a drug, a toxin, a ligand, a detection probe, and the like) has a specific function or activity. The disclosure also relates to methods for preparing the conjugate. The disclosure further relates to methods of using the conjugate to deliver an active agent to a target cell in a subject, as well as methods for treating a subject in need of the active agent (e.g., a subject having cancer).
(b) Background Art
Methods for inhibiting growth of cancer cells by targeted delivery of anti-cancer agents have been proposed. For example, it has been shown that targeted delivery of an antibody-drug conjugate can kill a particular cancer cell. As the antibody (or antibody fragment) specifically binds the cancer cell, the drug is delivered to the target cancer cell. Targeted delivery of the drug ensures that the drug acts on the target cancer cell instead of normal host cells, thereby minimizing the side effects resulting from damage to normal cells.
Antibody conjugates can be used to deliver chemical and/or biological molecules. Exemplary chemical and/or biological molecules include a drug conventionally used in chemical treatment, a bacterial protein toxin (e.g., diphtheria toxin), a plant protein toxin (e.g., ricin), a small molecule toxin (e.g., auristatin, geldanamycin, maytansinoid, calicheamycin, daunomycin, methotrexate, vindesine, and tubulysin), an affinity ligand, a detection probe (e.g., fluorescent probe, radioactive probe), and the like (including combinations thereof).
Antibody-drug conjugates that have been proposed thus far are prepared by bonding a drug moiety with a plurality of lysine groups of an antibody. Alternatively, antibody-drug conjugates are prepared by reducing all or part of the interchain disulfide groups of an antibody or reducing all the interchain disulfide groups followed by partial oxidation to thereby give free cysteine thiol groups, and then bonding the free cysteine thiol groups with a drug moiety.
Existing preparation methods, however, have some problems. For example, the overall preparation process is complicated because the antibody-drug conjugates prepared by the existing preparation methods are not uniform (homogeneous). When antibody-drug conjugates are prepared by bonding a drug moiety with lysine groups, various types and forms of antibody-drug conjugates are obtained due to the presence of many lysine groups in the antibody (e.g., 100 lysine groups per antibody). Similarly, when preparing antibody-drug conjugates by bonding thiol groups with a drug moiety, a mixture of diastereomers is obtained due to bonding between thiol groups and maleimide groups. For example, if n drugs are conjugated, a mixture of 2n stereoisomers is obtained. Thus, where the drug distribution number is 0-8 (e.g., where interchain disulfide groups are reduced), a mixture of
      ∑          n      =      0              n      =      8        ⁢          ⁢      2    n  of stereoisomers is obtained. In addition, where i drugs are conjugated with q sites, a mixture of
      ∑          i      =      0        q    ⁢          ⁢  qCiof different compounds is obtained.
Furthermore, when preparing antibody-drug conjugates by bonding lysine groups with a drug moiety, the electric charge of the lysine groups may be lost, thereby causing the antibody to lose its unique antigen specificity. Likewise, the tertiary or quaternary structure of the antibody may not be maintained when preparing antibody-drug conjugates by reducing disulfide groups, thereby causing the antibody to be inactivated or become a non-specific antibody. When preparing antibody-drug conjugates by using thiol-maleimide bonding, the drug may be cleaved (non-specifically) from the conjugates via, e.g., a reverse reaction.
To overcome the problems associated with the prior preparation methods, an alternative method was proposed in which amino acid groups in particular positions of an antibody are replaced with cysteine groups. Although this method shows better result than the prior preparation methods in terms of toxicity, activity, and safety, this method still involves thiol-maleimide bonding and thus suffers from the diastereomer and instability problems associated with thiol-maleimide bonding. Another alternative method was proposed in which selenocysteine groups are attached to the carboxy terminals of an antibody.
In addition to use of cysteine substitutions to control the site of conjugation, Ambrx Technology (at the World Wide Web (www) ambrx.com) has been working toward incorporating non-natural amino acids in the antibody to provide functional groups that can be used for linker chemistry. Ambrx's expression systems contain tRNA synthetases that aminoacylate the original tRNA with a non-natural amino acid, thereby inserting a non-natural amino acid whenever the amber stop is encountered.
Redwood Bioscience's (at the World Wide Web (www) redwoodbioscience.com) technology employs genetically encoded aldehyde tags and aims to exploit a specific sequence that is posttranslationally recognized and modified by an enzyme, i.e., a formyl glycine-generating enzyme, to produce a so-called aldehyde chemical handle. The incorporation of a CxPxR sequence at specific positions in the antibody provides a means to produce a reactive aldehyde amenable to drug conjugation.
However, in view of the above-mentioned problems in the art pertaining to making antibody-drug conjugates, new antibody-drug conjugates and new methods of making antibody-drug conjugates are highly desirable.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art.